The FoF1 ATP synthase of mitochondria, chloroplasts and bacteria couples the energy of the proton motive force to the synthesis of ATP. In the case of bacteria such as Escherichia coli, the enzyme also acts as an ATP-driven proton pump. Studies of the multiple subunit complex have shown that catalysis occurs in the soluble F1 sector while transport is carried out in the membranous Fo; however, very little is known about the how these two types of activities are linked. The long-range goal of this proposal is to understand the molecular mechanism of coupling between transport functions. To do this, the main tool will be the genetic technique of second-site reversion. Recently, several second- site mutations near the carboxyl terminus of the E. coli gamma subunit were found to suppress the effect of an amino terminal mutation, gammaMet-23-Lys, which caused extremely inefficient coupling. Because the second-site mutations were identified based on a functional selection, the suppressors in turn became implicated in coupling. Furthermore, because linkage between catalysis and transport must involve several subunits, regions of the gamma subunit defined by the suppressors should interact with others subunits. The first specific aim will seek to identify residues in those subunits by isolating a series of suppressor mutations. Taken together, a chain of interacting residues will define segments of the complex that participate in coupling. The second specific aim will take advantage of transport and coupling mutations to characterize how transport regulates catalysis. For the first time, kinetic parameters of partial reactions of these mutant enzymes will be measured to elucidate the catalytic steps which are coupled to transport. The third specific aim will obtain structural information which is critical for understanding the mechanism of coupling. The proximal position of residues suggested by the suppressor studies will be tested by replacing them with cysteine and assayed for formation of disulfide bonds. The cysteine mutations will also be useful for specific placement of spectroscopic probes to detect changes in conformation during enzyme activity. Finally, long-range plans include crystallization of F1 subunits for X-ray diffraction and structure determination so that the perturbations of mutations affecting coupling can be assessed within the protein structure. Each specific aim will contribute equally towards understanding how catalysis is coupled to transport.